2. Jakub Kostal & Steve Sontum Thesis Presentation ‘06

3. Courtesy of www.mcsrr.org

6. O 2 /CO Binding of CO/O 2 to Fe in Heme Fe N N N N CO ligand

7. Process of Ligand binding in Heme www.chemistry.wustl.edu

8. Myoglobin’s ability to bind oxygen is readily poisoned by its stronger affinity for carbon monoxide The affinity for CO is greatly reduced compared to free heme How does ligand surroundings in myoglobin’s Heme pocket influence ligand binding? 1. Sterics Bound vs. free Heme: The Ultimate Puzzle 2. Electrostatic interactions

9. Heme Pocket for Dummies

10. Studying Electrostatic Effects: Vibration of CO bond Triple bond character causes high vibration stretching frequencies (CO) used to characterize different conformers of the bound state Equilibrium IR absorbance spectrum of bound CO shows the major sub states A 0, A 1 and A 3 are associated with CO stretching bands at 1966, 1945, and 1927cm -1 Dispersion of A sub states thought to be caused by electrostatic interactions between the CO dipole and the imidazole chain of the distal His64, which assumes different dynamic conformations

11. Studying Electrostatic Effects: Vibration of CO bond Triple bond character causes high vibration stretching frequencies (CO) used to characterize different conformers of the bound state Equilibrium IR absorbance spectrum of bound CO shows the major sub states A 0, A 1 and A 3 are associated with CO stretching bands at 1966, 1945, and 1927cm -1 Dispersion of A sub states thought to be caused by electrostatic interactions between the CO dipole and the imidazole chain of the distal His64, which assumes different dynamic conformations

12. Models for electrostatic interactions in the CO complexes - amino acid mutations A (A 3 ): Asn68 Mb ( CO = 1938cm -1 ; Fe-C = 527cm -1 ) B (A 1, A 2 ): Wild-Type Mb ( CO = 1945cm -1 ; Fe-C = 507cm -1 ) C (A 0 ): Val64/Thr68 Mb ( CO = 1984cm -1 ; Fe-C = 477cm -1 )

13. Preliminary studies on our project: Building a simple Theoretical Model Generation of a vibrational force field using RESP method for various heme analogs with bound CO ligand Classical MD model built Dynamic Simulation of an out-of-plane electric field using Li ions to predict changes in CO vibrations based on experimental observations Hypothesis: Li (+/-) Li (-/+) (CO) < (CO) Molecular Dynamics Trajectory

14. Observed Vibrational Shifts Expected trends somewhat preserved only at high e. field intensities

15. “Torquing” motion of the CO ligand Fe-C-O bond locked in one “torquing” mode throughout the dynamic trajectory at higher E. field intensities Dominant mode at higher E. field Torquing motion accounts for additional centripetal stretching of the CO bond

16. Analysis of the torquing mode Fe-O angle to the normal (  ) is greater than Fe-C angle to the normal of the heme plane (  ). This differences increases with reversed e. field Direction of the electric field changes  and  As the intensity of e. field increases,  and  increase as well As the temperature increases, both angles increase Normal direction Reversed direction X-axis: time (0.01ps) Y-axis: angle (degree)

17. MD Trajectory of Full Myoglobin NN NN Out of heme pocket Inside heme pocket

18. Conclusions and Future Work We have successfully generated RESP force field for CO heme model to study the effect of electrostatic fields on the vibration of CO. We have observed a toquing motion of the CO ligand induced by electrostatic fields of high intensities. We have analyzed 2ns MD trajectory of full myoglobin and observed that distal His64 spends 88% of the time inside and 12% outside of the heme pocket. Generate force fields for similar O-O and NO bound heme models (in progress)

19. Acknowledgments Steve Meghan Judy

20. References Spiro T. G., Kozlowski P. M. 1998. Discordant results on FeCO deformability in heme proteins reconciled by density functional theory. J. Am. Chem. Soc. 120: 4524-4525 Phillips G. N., Teodoro M. L., Tiansheng L., Smith B., Olson J. S.. 1999. Bound CO Is a Molecular Probe of Electrostatic Potential in the Distal Pocket of Myoglobin. J. Phys. Chem. B. 103: 8817-8829 Nienhaus K., Pengchi D., Kriegl J. M., Nienhaus G. U. 2003. Structural Dynamics of Myoglobin: Effect of Internal Cavities on Ligand Migration and Binding. Biochemistry. 42: 9647-9658 Ray, G. B., X.-Y. Li, J. A. Ibers, J. L. Sessler, and T. G. Spiro. 1994. How far proteins bend the FeCO unit? Distal polar and steric effects in heme proteins and models. J. Am. Chem. Soc. 116: 162-176. Rovira, C., K. Kunc, J. Hutter, P. Ballone, and M. Parrinello. 1997. Equilibrium geometries and electronic structure of iron-porphyrin complexes: a density functional study. J. Phys. Chem. A. 101:8914-8925.

22. Solvated WT Trajectory